Effects of Salinity and Abscisic Acid on Lipid Transfer Protein Accumulation, Suberin Deposition and Hydraulic Conductance in Pea Roots.

Lipid transfer proteins (LTPs) participate in many important physiological processes in plants, including adaptation to stressors, e.g., salinity. Here we address the mechanism of this protective action of LTPs by studying the interaction between LTPs and abscisic acid (ABA, a "stress" hormone) and their mutual participation in suberin deposition in root endodermis of salt-stressed pea plants. Using immunohistochemistry we show for the first time NaCl induced accumulation of LTPs and ABA in the cell walls of phloem paralleled by suberin deposition in the endoderm region of pea roots. Unlike LTPs which were found localized around phloem cells, ABA was also present within phloem cells. In addition, ABA treatment resulted in both LTP and ABA accumulation in phloem cells and promoted root suberization. These results suggested the importance of NaCl-induced accumulation of ABA in increasing the abundance of LTPs and of suberin. Using molecular modeling and fluorescence spectroscopy we confirmed the ability of different plant LTPs, including pea Ps-LTP1, to bind ABA. We therefore hypothesize an involvement of plant LTPs in ABA transport (unloading from phloem) as part of the salinity adaptation mechanism.

Rhizobacteria Inoculation Effects on Phytohormone Status of Potato Microclones Cultivated In Vitro under Osmotic Stress.

Water deficits inhibit plant growth and decrease crop productivity. Remedies are needed to counter this increasingly urgent problem in practical farming. One possible approach is to utilize rhizobacteria known to increase plant resistance to abiotic and other stresses. We therefore studied the effects of inoculating the culture medium of potato microplants grown in vitro with Azospirillum brasilense Sp245 or Ochrobactrum cytisi IPA7.2. Growth and hormone content of the plants were evaluated under stress-free conditions and under a water deficit imposed with polyethylene glycol (PEG 6000). Inoculation with either bacterium promoted the growth in terms of leaf mass accumulation. The effects were associated with increased concentrations of auxin and cytokinin hormones in the leaves and stems and with suppression of an increase in the leaf abscisic acid that PEG treatment otherwise promoted in the potato microplants. O. cytisi IPA7.2 had a greater growth-stimulating effect than A. brasilense Sp245 on stressed plants, while A. brasilense Sp245 was more effective in unstressed plants. The effects were likely to be the result of changes to the plant's hormonal balance brought about by the bacteria.

Role of Pea LTPs and Abscisic Acid in Salt-Stressed Roots.

Lipid transfer proteins (LTPs) are a class of small, cationic proteins that bind and transfer lipids and play an important role in plant defense. However, their precise biological role in plants under adverse conditions including salinity and possible regulation by stress hormone abscisic acid (ABA) remains unknown. In this work, we studied the localization of LTPs and ABA in the roots of pea plants using specific antibodies. Presence of LTPs was detected on the periphery of the cells mainly located in the phloem. Mild salt stress (50 mM NaCI) led to slowing plant growth and higher immunostaining for LTPs in the phloem. The deposition of suberin in Casparian bands located in the endoderma revealed with Sudan III was shown to be more intensive under salt stress and coincided with the increased LTP staining. All obtained data suggest possible functions of LTPs in pea roots. We assume that these proteins can participate in stress-induced pea root suberization or in transport of phloem lipid molecules. Salt stress increased ABA immunostaining in pea root cells but its localization was different from that of the LTPs. Thus, we failed to confirm the hypothesis regarding the direct influence of ABA on the level of LTPs in the salt-stressed root cells.

Study of cytokinin transport from shoots to roots of wheat plants is informed by a novel method of differential localization of free cytokinin bases or their ribosylated forms by means of their specific fixation.

The aim of the present report was to demonstrate how a novel approach for immunohistochemical localization of cytokinins in the leaf and particularly in the phloem may complement to the study of their long-distance transport. Different procedures of fixation were used to conjugate either cytokinin bases or their ribosides to proteins of cytoplasm to enable visualization and differential localization of these cytokinins in the leaf cells of wheat plants. In parallel to immunolocalization of cytokinins in the leaf cells, we immunoassayed distribution of free bases of cytokinins, their nucleotides and ribosides between roots and shoots of wheat plants as well as their presence in phloem sap after incubation of leaves in a solution supplemented with either trans-zeatin or isopentenyladenine. The obtained data show ribosylation of the zeatin applied to the leaves and its elevated level in the phloem sap supported by in vivo localization showing the presence of ribosylated forms of zeatin in leaf vessels. This suggests that conversion of zeatin to its riboside is important for the shoot-to-root transport of zeatin-type cytokinins in wheat. Exogenous isopentenyladenine was not modified, but diffused from the leaves as free base. These metabolic differences may not be universal and may depend on the plant species and age. Although the measurements of cytokinins in the phloem sap and root tissue is the most defining for determining cytokinin transport, study of immunolocalization of either free cytokinin bases or their ribosylated forms may be a valuable source of information for predicting their transport in the phloem and to the roots.

Rapid changes in root HvPIP2;2 aquaporins abundance and ABA concentration are required to enhance root hydraulic conductivity and maintain leaf water potential in response to increased evaporative demand.

To address the involvement of abscisic acid (ABA) in regulating transpiration and root hydraulic conductivity (LpRoot) and their relative importance for maintaining leaf hydration, the ABA-deficient barley mutant Az34 and its parental wild-type (WT) genotype (cv. Steptoe) were grown in hydroponics and exposed to changes in atmospheric vapour pressure deficit (VPD) imposed by air warming. WT plants were capable of maintaining leaf water potential (ψL) that was likely due to increased LpRoot enabling higher water flow from the roots, which increased in response to air warming. The increased LpRoot and immunostaining for HvPIP2;2 aquaporins (AQPs) correlated with increased root ABA content of WT plants when exposed to increased air temperature. The failure of Az34 to maintain ψL during air warming may be due to lower LpRoot than WT plants, and an inability to respond to changes in air temperature. The correlation between root ABA content and LpRoot was further supported by increased root hydraulic conductivity in both genotypes when treated with exogenous ABA (10-5 M). Thus the ability of the root system to rapidly regulate ABA levels (and thence aquaporin abundance and hydraulic conductivity) seems important to maintain leaf hydration.

Dependence of growth inhibiting action of increased planting density on capacity of lettuce plants to synthesize ABA.

Inhibition of lettuce plant growth under increased planting density was accompanied by accumulation of abscisic acid (ABA) in the shoots of competing plants. To check causal relationship between these responses we studied the effect of decreased synthesis of ABA on growth indexes and hormonal balance of lettuce plants under elevated density of their planting (one (single) or three (competing) plants per pot). Herbicide fluridone was used to inhibit ABA synthesis. Preliminary experiments with single plants showed that presence of fluridone in the soil solution at rather low concentration (0.001mg/L) did not affect either chlorophyll content or growth rate of shoots and roots during at least one week. Treatment of competing (grouped) plants with this concentration of fluridone prevented both accumulation of ABA and competition induced growth inhibition. These results confirm important role of this hormone in the growth inhibiting effect of increased planting density. Furthermore, as in the case of ABA, fluridone prevented allocation of indoleacetic acid (IAA) to the shoots of competing plants likely contributing to leveling off the increase in the ratio of leaf area to their mass that is characteristic effect of shading in the dense plant populations. The results suggest involvement of ABA in allocation of IAA in competing plants. Application of fluridone did not influence the concentration of cytokinins in the shoots, whose level was decreased by competition either in fluridone treated or control (untreated with fluridone) plants. Accumulation of ABA in the shoots of competing plants accompanied by inhibition of their growth and the absence of either accumulation of ABA or inhibition of their growth in fluridone treated grouped plants confirms importance of ABA synthesis for growth response to competition.

Common and specific responses to availability of mineral nutrients and water.

Changes in resource (mineral nutrients and water) availability, due to their heterogeneous distribution in space and time, affect plant development. Plants need to sense these changes to optimize growth and biomass allocation by integrating root and shoot growth. Since a limited supply of water or nutrients can elicit similar physiological responses (the relative activation of root growth at the expense of shoot growth), similar underlying mechanisms may affect perception and acquisition of either nutrients or water. This review compares root and shoot responses to availability of different macronutrients and water. Attention is given to the roles of root-to-shoot signalling and shoot-to-root signalling, with regard to coordinating changes in root and shoot growth and development. Involvement of plant hormones in regulating physiological responses such as stomatal and hydraulic conductance is revealed by measuring the effects of resource availability on phytohormone concentrations in roots and shoots, and their flow between roots and shoots in xylem and phloem saps. More specific evidence can be obtained by measuring the physiological responses of genotypes with altered hormone responses or concentrations. We discuss the similarity and diversity of changes in shoot growth, allocation to root growth, and root architecture under changes in water, nitrate, and phosphorus availability, and the possible involvement of abscisic acid, indole-acetic acid, and cytokinin in their regulation. A better understanding of these mechanisms may contribute to better crop management for efficient use of these resources and to selecting crops for improved performance under suboptimal soil conditions.

Cytokinin producing bacteria stimulate amino acid deposition by wheat roots.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.

Accumulation of cytokinins in roots and their export to the shoots of durum wheat plants treated with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP).

Cytokinin flow from roots to shoots can serve as a long-distance signal important for root-to-shoot communication. In the past, changes in cytokinin flow from roots to shoots have been mainly attributed to changes in the rate of synthesis or breakdown in the roots. The present research tested the possibility that active uptake of cytokinin by root cells may also influence its export to shoots. To this end, we collapsed the proton gradient across root membranes using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit secondary active uptake of exogenous and endogenous cytokinins. We report the impact of CCCP on cytokinin concentrations and delivery in xylem sap and on accumulation in shoots of 7-day-old wheat plants in the presence and absence of exogenous cytokinin applied as zeatin. Zeatin treatment increased the total accumulation of cytokinin in roots and shoots but the effect was smaller for the shoots. Immunohistochemical localization of cytokinins using zeatin-specific antibodies showed an increase in immunostaining of the cells adjacent to xylem in the roots of zeatin-treated plants. Inhibition of secondary active cytokinin uptake by CCCP application decreased cytokinin accumulation in root cells but increased both flow from the roots and accumulation in the shoots. The possible importance of secondary active uptake of cytokinins by root cells for the control of their export to the shoot is discussed.

Plant hormone interactions: innovative targets for crop breeding and management.

Here we highlight how both the root and shoot environment impact on whole plant hormone balance, particularly under stresses such as soil drying, and relate hormone ratios and relative abundances to processes influencing plant performance and yield under both mild and more severe stress. We discuss evidence (i) that abscisic acid (ABA) and ethylene act antagonistically on grain-filling rate amongst other yield-impacting processes; (ii) that ABA's effectiveness as an agent of stomatal closure can be modulated by coincident ethylene or cytokinin accumulation; and (iii) that enhanced cytokinin production can increase growth and yield by improving foliar stay-green indices under stress, and by improving processes that impact grain-filling and number, and that this can be the result of altered relative abundances of cytokinin and ABA (and other hormones). We describe evidence and novel processes whereby these phenomena are/could be amenable to manipulation through genetic and management routes, such that plant performance and yield can be improved. We explore the possibility that a range of ABA-ethylene and ABA-cytokinin relative abundances could represent targets for breeding/managing for yield resilience under a spectrum of stress levels between severe and mild, and could circumvent some of the pitfalls so far encountered in the massive research effort towards breeding for increases in the complex trait of yield.

Effect on shoot water relations, and cytokinin and abscisic acid levels of inducing expression of a gene coding for isopentenyltransferase in roots of transgenic tobacco plants.

Heat shock (HS) at 40 degrees C was given to the root system of Nicotiana tabacum wild type (WT) and to HSIPT transgenic plants transformed with the bacterial cytokinin biosynthesis gene isopentenyltransferase (ipt) cloned behind the heat shock 70 promoter from Drosophila melanogaster. HS increased cytokinin concentrations in roots and leaves of transgenic plants. The effect was smaller in WT plants and restricted to upper leaves. HS also increased the activity of the cytokinin-degrading enzyme cytokinin oxidase in leaves of transgenic plants. This suggests that increases in cytokinin concentration induced by HS were lessened but not eliminated by increases in cytokinin oxidase. Elevated levels of zeatin riboside (the main transportable form of cytokinin) were also found in the HS-treated roots. It is proposed that increases in leaves were the outcome of increased transport of this hormone from roots in the transpiration stream. In conjunction with increased leaf cytokinin concentration, HS treatment to the roots increased stomatal conductivity and transpiration in both transgenic and WT plants. Subsequently, increased transpiration depressed leaf relative water content. This, in turn, raised leaf abscisic acid (ABA) concentrations, resulting in stomatal closure. It is concluded that the preceding increases in leaf cytokinin concentration, stomatal opening, and faster transpiration resulting from the localized induction of ip gene expression in roots strengthens the concept of cytokinin involvement in root to shoot signalling.

ABA mediation of shoot cytokinin oxidase activity: assessing its impacts on cytokinin status and biomass allocation of nutrient-deprived durum wheat.

Although nutrient deprivation alters the concentrations of several plant hormones, the role of each in decreasing shoot-to-root ratio is not clear. A 10-fold dilution of the nutrient concentration supplied to hydroponically-grown 7-day-old durum wheat (Triticum turgidum L. ssp. durum Desf.) plants decreased shoot growth, shoot-to-root ratio and shoot and root cytokinin concentrations, increased shoot ABA concentration and shoot cytokinin oxidase activity, but had no effect on xylem sap ABA and cytokinin concentrations. Nutrient deprivation also increased xylem concentrations of conjugated ABA. The role of ABA in these responses was addressed by adding 11.4 µm ABA to the nutrient solution of well fertilised plants, or 1.2 mm fluridone (an inhibitor of ABA biosynthesis) to the nutrient solution of nutrient-deprived plants. The former induced similar changes in shoot-to-root ratio (by inhibiting shoot growth), shoot ABA concentration, shoot and root cytokinin concentrations and shoot cytokinin oxidase activity as nutrient deprivation. Conversely, fluridone addition to nutrient-deprived plants restored shoot-to-root ratio (by inhibiting root growth), shoot ABA concentration, shoot and root cytokinin concentrations to levels similar to well fertilised plants. Although root growth maintenance during nutrient deprivation depends on a threshold ABA concentration, shoot growth inhibition is independent of shoot ABA status. Although fluridone decreased shoot cytokinin oxidase activity of nutrient-deprived plants, it was still 1.7-fold greater than well fertilised plants, implying that nutrient deprivation could also activate shoot cytokinin oxidase independently of ABA. These data question the root signal basis of cytokinin action, but demonstrate that changes in ABA status can regulate shoot cytokinin concentrations via altering their metabolism.

Abscisic acid accumulation in the roots of nutrient-limited plants: its impact on the differential growth of roots and shoots.

We describe the involvement of abscisic acid (ABA) in the control of differential growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold dilution of the optimal concentration of nutrient solution inhibited shoot growth, while root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation in favour of the roots. This suggests the involvement of ABA in the redirecting of growth in favour of roots under limited nutrient supply. The ABA content was greater in shoots and growing apical root parts of starved plants than in nutrient sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem apparently contributed to the accumulation of ABA in the apical part of the roots. Thus, partitioning of growth between roots and shoots of wheat plants limited in mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA may originate in the shoots, where its synthesis is stimulated by the loss of leaf turgor.

Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings and its putative function.

The distribution pattern of a 70 kDa cytokinin-binding protein (CBP70) was studied in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus). CBP70 was detected in crude protein extracts of all root zones and shoot parts by western blotting and by the sandwich ELISA (enzyme-linked immunosorbent assay) technique, using a pair of monoclonal anti-CBP70 antibodies cross-reacting with non-overlapping protein epitopes. The highest amount of CBP70 was found in the root meristem, which corresponds to the concentration in the meristem of zeatin, its riboside, nucleotide, and 9N-glucoside. CBP70 accumulation was also detected in other zones of cell division: in the root cap, shoot apex, and vascular tissues, suggesting involvement of the protein in the processes related to cell proliferation. This suggestion was also supported by CBP70 distribution in the root meristem: mitotically inactive cells of the quiescent centre did not contain a detectable amount of the protein. Stem cells adjoining the quiescent centre contained less CBP70 than their daughter cells. Using monoclonal antibodies against CBP70 for immunocytochemistry, the presence of the protein in the cytoplasm and its accumulation in nuclei and especially in nucleoli was demonstrated; such a pattern was observed in all cell types of seedlings. The subcellular distribution pattern of CBP70 was analysed by immunogold electron microscopy of the meristem and leaf cells; CBP70 was localized in the cytoplasm and nucleoplasm, and its highest concentration was detected in nucleoli. CBP70 was not detected in the vacuole and cell wall. In the RNA polymerase I model system, purified CBP70 mediated a trans-zeatin-dependent activation of transcription in vitro, and anti-CBP70 monoclonal antibodies blocked this activation. Other natural and synthetic physiologically active cytokinins also activated transcript elongation in the model system in the presence of CBP70. Adenine and inactive analogues of cytokinins had no such effects. These data suggest that CBP70 is a transcript elongation factor or a modulator of elongation factor activity specifically mediating a cytokinin-dependent regulation of transcription.

Effect of partial rootzone drying on the concentration of zeatin-type cytokinins in tomato (Solanum lycopersicum L.) xylem sap and leaves.

Decreased cytokinin (CK) export from roots in drying soil might provide a root-to-shoot signal impacting on shoot physiology. Although several studies show that soil drying decreases the CK concentration of xylem sap collected from the roots, it is not known whether this alters xylem CK concentration ([CK(xyl)]) in the leaves and bulk leaf CK concentration. Tomato (Solanum lycopersicum L.) plants were grown with roots split between two soil columns. During experiments, water was applied to both columns (well-watered; WW) or one (partial rootzone drying; PRD) column. Irrigation of WW plants aimed to replace transpirational losses every day, while PRD plants received half this amount. Xylem sap was collected by pressurizing detached leaves using a Scholander pressure chamber, and zeatin-type CKs were immunoassayed using specific antibodies raised against zeatin riboside after separating their different forms (free zeatin, its riboside, and nucleotide) by thin-layer chromatography. PRD decreased the whole plant transpiration rate by 22% and leaf water potential by 0.08 MPa, and increased xylem abscisic acid (ABA) concentration 2.5-fold. Although PRD caused no detectable change in [CK(xyl)], it decreased the CK concentration of fully expanded leaves by 46%. That [CK(xyl)] was maintained and not increased while transpiration decreased suggests that loading of CK into the xylem was also decreased as the soil dried. That leaf CK concentration did not decline proportionally with CK delivery suggests that other mechanisms such as CK metabolism influence leaf CK status of PRD plants. The causes and consequences of decreased shoot CK status are discussed.

Fast changes in expression of expansin gene and leaf extensibility in osmotically stressed maize plants.

Adding PEG to the nutrient medium of maize (Zea mays L., hybrid Harkovskaya 310 MV) plants arrested the growth of their leaves initially but in 40-50 min growth resumed. This coincided with and was obviously due to a gradual increase in extensibility of the primary leaf suggested by changes in its extension rate, which was induced by adding a counterweight to inductive electromechanical position sensor. Specificity of gene probe for expansins was confirmed by sequencing cDNA and its comparison with literature data. Dot-blot analysis showed an increase in transcript level of expansin genes induced by PEG treatment. Thus gene-specific regulation of expansin mRNA pools likely contributes to fast adjustment of cell wall-loosening under conditions of water deficit.

The effect of root cooling on hormone content, leaf conductance and root hydraulic conductivity of durum wheat seedlings (Triticum durum L.).

Root cooling of 7-day-old wheat seedlings decreased root hydraulic conductivity causing a gradual loss of relative water content during 45 min (RWC). Subsequently (in 60 min), RWC became partially restored due to a decrease in transpiration linked to lower stomatal conductivity. The decrease in stomatal conductivity cannot be attributed to ABA-induced stomatal closure, since no increase in ABA content in the leaves or in the concentration in xylem sap or delivery of ABA from roots was found. However, decreased stomatal conductance was associated with a sharp decline in the content of cytokinins in shoots that was registered shortly after the start of root cooling and linked to increases in the activity of cytokinin-oxidase. This decrease in shoot cytokinin content may have been responsible for closing stomata, since this hormone is known to maintain stomatal opening when applied to plants. In support of this, pre-treatment with synthetic cytokinin benzyladenine was found to increase transpiration of wheat seedlings with cooled roots and bring about visible loss of turgor and wilting.

Effect of partial root excision on transpiration, root hydraulic conductance and leaf growth in wheat seedlings.

Removal of four out of five roots did not lower transpiration and stomatal conductivity of wheat (Triticum durum Desf.) seedlings. Water content of mature expanded leaf lamina remained constant at control levels. The results suggest that the only remaining root was capable to supply the shoot with water. This was evidenced by an increase in hydraulic conductivity of the root system following partial root excision measured at low subatmospheric pressures induced by vacuum. In the absence of a hydrostatic gradient, water flow from reduced root system was initially not higher than from an intact system, but increased subsequently. ABA content was increased in roots 1 h after partial root excision, which might contribute to the increase in hydraulic conductivity.

Effect of partial root excision on shoot water relations.

Removing 4 out of 5 serminal roots from 7-day-old wheat seedlings arrested leaf elongation for 1.5 h. This effect can be explained by an initial decrease in foliar water content resulting from the smaller root surface area available for water uptake. Subsequently, leaf hydration increased with time and came to equal that of intact plants within 2 h. The rehydration was seemingly effected by an increasing conductivity of the one remaining root axis, since transpiration of the partially de-rooted plants did not fall below that of controls. With time, leaf elongation resumed, but at a slower rate than in intact plants. This slower growth may be attributed to a decrease in leaf extensibility since this was found to be reduced when measured by a counterweight technique involving linear displacement transducers. Loss of extensibility was associated with decreased IAA concentration in the leaf elongation zone.